Project Summary Susceptibility to Zika virus (ZIKV) infection is highly variable. While most seropositive individuals remain asymptomatic, approximately 18% develop symptoms that range from a mild rash in the majority of cases to Guillain-Barr syndrome or microcephaly in the minority (less than 1% of neonates experience severe pathology). Like all known vector-borne flaviviruses, ZIKV must evade type I interferon (IFN) signaling to establish infection. IFNs are cytokines secreted by host cells in response to a pathogen that trigger the establishment of an antiviral state in infected and neighboring cells via JAK/STAT-mediated transactivation of over 400 IFN stimulated genes (ISGs). Recent evidence points strongly towards the degradation of the ISG, signal transducer and activator of transcription 2 (STAT2) by non-structural protein 5 (NS5) as a putative mechanism by which ZIKV escapes IFN, thereby implicating STAT2 as a dominant restriction factor in ZIKV infection. As STAT2 is an essential component of the type I IFN signaling pathway, its removal abolishes all canonical induction of ISGs. Notably, mice are resistant to both ZIKV and closely related dengue virus as their NS5s do not inhibit murine STAT2, but STAT2-defcient mice are highly susceptible. This suggests that factors that affect the ability of NS5 to clear host STAT2 impact disease outcome following infection. Our group has previously identified rare germline mutations that confer resistance to a broad spectrum of viral infections. Individuals deficient for ubiquitin specific peptidase 18 (USP18), a negative regulator of IFN, or for IFN stimulated gene 15 (ISG15), a ubiquitin-like peptide that stabilizes USP18, exhibit elevated STAT2 levels for up to 6 days post exposure to IFN. Dermal fibroblasts derived from ISG15-/- and USP18-/- individuals showed 1,000 ? 100,000 fold stronger inhibition of ZIKV replication when pretreated with IFN?-2b. We also identified genetic variations in regions of STAT2 that likely alter the STAT2-NS5 interaction based on in silico damage prediction, species specificity of NS5 for human, but not murine, STAT2, and common targeting of STAT2 by NS5 from related flaviviruses. We therefore suggest that elevated STAT2 levels in ISG15-/- and USP18-/- cells inhibit ZIKV by creating a stoichiometric imbalance that favors IFN signaling despite NS5- mediated degradation of STAT2, and that heterogeneity within the STAT2 gene can explain differing levels of disease.